Compositions including ammonia oxidizing bacteria and methods of using same

ABSTRACT

A method of enhancing health through the generation in close proximity of a surface of a subject, nitric oxide and nitric oxide precursors using bacteria adapted to oxidize ammonia and urea derived from perspiration is described. Local and systemic effects are described including reduction in vascular disease, enhancement of sexual function, improved skin health, and reduced transmission of sexually transmitted diseases.

This application claims priority to U.S. Provisional Application Ser.No. 60/224,598 filed Aug. 11, 2000.

FIELD OF INVENTION

The present invention relates to a composition including ammoniaoxidizing bacteria to increase production of nitric oxide andnitric-oxide precursors on the surface of a subject and methods of usingsame.

BACKGROUND

Beneficial bacteria have been utilized to suppress the growth ofpathogenic bacteria. Bacteria and other microorganisms are ubiquitous inthe environment. The discovery of pathogenic bacteria and the germtheory of disease has had a tremendous effect on health and diseasestates. Bacteria are a normal part of the intestinal contents of allliving things. These bacteria are not pathogenic under normalconditions, and in fact improve health by rendering the normalintestinal contents less hospitable for disease causing organisms. Thisis accomplished in a number of ways: nutrients are consumed, leavingless for pathogens; conditions are produced, such as pH, oxygen tension,which are not hospitable for pathogens; compounds are produced that aretoxic to pathogens; pathogens are consumed as food by thesemicroorganisms; less physical space remains available for pathogens; andspecific binding sites are occupied leaving fewer for pathogens. Thepresence of these desirable bacteria is seen as useful in preventingdisease states.

Fermentation of food products has been done to substitute a desirednon-pathogenic strain for potential spoilage or pathogenic organisms.Brewed beverages, wine, pickled food, fermented milk products includingcheese, yogurt, buttermilk, sausage are all examples where desiredmicroorganisms are deliberately inoculated into food products underconditions that favor their growth and inhibit the growth of spoilageand pathogenic strains. U.S. patents disclosing the use of specificbacteria to inhibit the growth of harmful bacteria include: U.S. Pat.No. 3,984,575 issued to Farr Oct. 5, 1976; U.S. Pat. No. 4,689,226issued to Nurmi, et al. Aug. 25, 1987; U.S. Pat. No. 5,322,686 issued toGrahn, et al. June 21, 1994; U.S. Pat. No. 5,451,400 issued to Stern, etal. Sep. 19, 1995; U.S. Pat. No. 5,604,127 issued to Nisbet, et al. Feb.18, 1997; and U.S. Pat. No. 5,807,546 issued to Stern, et al. Sep. 15,1998.

U.S. Pat. No. 5,176,911 issued to Tosi, et al. Jan. 5, 1993 disclosesthe use of specific bacteria recovered from healthy asymptomaticpatients and characterized in the laboratory as a preventative andcurative topical application to the vaginal area of women suffering fromvaginal yeast infections.

U.S. Pat. No. 5,534,253 issued to Casas, et al. Jul. 9, 1996, disclosesadministering a specific Lactobacillus reuteri strain through externalspraying, incorporation into feed, or injection into eggs, to produce anantibiotic substance identified as .beta.-hydroxypropionaldehyde.

U.S. Pat. No. 6,080,401 issued to Reddy, et al. Jun. 27, 2000, disclosesthe addition of various probiotics to herbal and pharmaceutical drugs toincrease their efficacy. The probiotic is selected from the groupconsisting of non-pathogenic members of genus Lactococcus,Lactobacilius, Pediococcus, Streptococcus, Propionibacterium,Brevibacterium, Penicillium, and Saccharomyces, and mixtures thereof. Inthe examples, the bacteria are ingested along with the drug or used in atooth cleaning preparation.

SUMMARY

The present invention relates to a method of supplying a nitric oxidecompound to a subject by positioning ammonia oxidizing bacteria in closeproximity to the surface of the subject.

Another embodiment of the present invention relates to an article ofclothing treated with bacteria adapted to metabolize any of ammonia,ammonium salts, or urea into nitric oxide and/or nitric oxideprecursors.

The present invention is also directed to a preparation to be applied toa surface of a subject comprising ammonia oxidizing bacteria adapted tometabolize any of ammonia, ammonium salts, or urea into nitric oxideand/or nitric oxide precursors.

DETAILED DESCRIPTION

The present invention relates to a to a composition including ammoniaoxidizing bacteria to increase production of nitric oxide and/or nitricoxide precursors in close proximity to a surface of a subject andmethods of using same. More specifically, applying a composition of anammonia oxidizing bacteria to skin during or after bathing to metabolizeurea and other components of perspiration into nitrite and ultimatelyinto Nitric Oxide (NO) results in a natural source of NO. One asp et ofthe present invention causes topical nitric oxide release at or near thesurface of the Skin where it can diffuse into the skin and have local aswell as systemic effects. This naturally produced nitric oxide can thenparticipate in the normal metabolic pathways by which nitric oxide isutilized by the body. Adding urea or ammonium salts to the skin providesadditional substrates that these bacteria utilize to form nitrite. Asused herein, the phrase near the surface is defined as adjacent to or inclose proximity to, but need not be in contact with the surface.

The invention is understood by realizing that until the advent ofrunning hot water and soap, bathing was infrequent. Under suchconditions (prevailing for >99.9% of historic and prehistoric time) theskin would develop a natural community of microorganisms adapted to theskin environment. An abundant component of human perspiration is urea.In soil, natural bacteria act upon urea and hydrolyze it to ammonia,which is then oxidized to nitrite, followed by rapid oxidation, by stillother bacteria, to nitrate. In soil, all nitrogen containing compoundsare ultimately degraded to nitrate. In fact it is nitrate that mostplants absorb as their nitrogen source. Under conditions of infrequentbathing, skin bacteria that can metabolize urea into nitrite wouldthrive and proliferate. The resulting nitrite on the skin when dampenedby additional perspiration at the normal sweat pH of 4.5 would releaseNO.

Nitric Oxide is a small molecule that diffuses rapidly trough the skininto the capillaries of the skin. Vasodilatation of these capillarieswould occur, as well as diffusion of NO into the blood where it may betransported to other regions of the body. Dilatation of the capillariesat the skin surface enhances blood flow to, and hence heat loss from,the skin during periods of exercise.

Heart disease and other vascular diseases are a significant cause ofdeath in the developed world. Vascular diseases also cause significantreductions in quality of life for those afflicted. Significant medicalresources are devoted to prevention, treatment and research into thecauses of these forms of disease.

Exercise has long been touted as having protective effects on the heart,the vascular system, and on health in general. Numerous studies andreports have shown an inverse correlation between exercise and deathfrom heart disease. Curiously the protective effects of exercise on thevascular system are sometimes seen to be lower at more vigorous activitylevels. This diminished protective effect of more vigorous physicalactivity is tot observed in all studies but has been observed for bothheart disease and stroke. A recent study, “Physical Activity and StrokeIncidence The Harvard Alumni Health Study”, by I-Min Lee, et al.(Stroke. 1998;29:2049-2054) showed a U shaped curve of stroke incidenceverses intensity of exercise. Walking was also observed to reduce strokeincidence independent of other forms of exercise. The authors wereunable to explain these observations, nor has a satisfactory explanationof these observations yet been made.

Death rates due to heart disease often show significant seasonalvariation. A recent article “Seasonal Variation in Chronic Heart FailureHospitalizations and Mortality in France”, Fabrice Boulay, MD, et al.(Circulation. 1999;100:280-286.) shows pronounced increases in mortalityduring the winter months and declines during the summer months over a 6year period. This study which covered the entire French population,showed a peak monthly average for January that was 20% above the yearlyaverage. The monthly minimum was 15% below the average in August. Thispattern is visible each and every year included in the study but nosatisfactory explanation for this data is provided.

Diet, smoking, exercise, control of high blood pressure, being married,personality type, genetic factors, viral infections, moderate alcoholconsumption have all been shown to affect rates of heart and vasculardiseases. With so many factors being important it is very difficult tofind the proper controls to correct for known as well as potentialunknown confounding factors. I have found that another factor, which iseasily controlled, may explain some of the discrepancy between differentrates of vascular disorders.

Physical activity induces a number of physiological changes. As physicalexertion increases, heart and respiration rate increase to supply fueland oxygen to the cells producing work. Since this production of work isnot 100% efficient, metabolic heat also increases and must bedissipated. The body increases sweat production to dissipate this heatthrough evaporative cooling. While Western medicine has focused on theprompt physiological effects of exercise, sweating per se also hasproponents. Raising the ambient temperature, as in a sauna, has beenclaimed to have salutary effects on one's heath. In fact the use of hightemperatures to induce sweating has been a common component of personalhygiene in many cultures prior to the introduction of soap and running(hot) water. The Turkish hammam, the Finnish sauna, the Native Americansweat lodger the Russian bania, and the Central American temascal areall examples of the use of high temperature for personal cleansing andhygiene. The Greeks and Roman baths are similar with written recordsdating from the fifth century BCE. The popular explanation for thehealth effects of sauna-like treatments has been the “flushing” oftoxins out of the body through increased sweating.

While modern medicine has had many advances in the understanding ofhuman physiology, there is still a great deal that remains unexplained.Traditional medicines and practices are often a useful source ofcompounds and procedures to test for medicinal properties. Thus a methodthat improves the body's natural ability to regulate and enhance theformation, and release of nitric oxide may have significant andwidespread health benefits.

Advances in understanding the interaction of nitric oxide with thephysiology of the human vascular system have been made. One advance waspthe discovery by Drs. Robert F. Furchgott, Louis J. Ignarro, and FeridMurad that Nitric Oxide is a vasodilator has paved the way forunderstanding the mechanism of action of organic nitrates, likenitroglycerine, as vasodilators, and for new drugs that increase thetime of action of endogenous NO, like Viagra.

Nitric oxide has also been implicated as a component of the human body'snatural defense against disease causing organisms. In a book, “NitricOxide and Infection”, Ferric C. Fang ed., Kluwer Academic/PlenumPublishers, 1999, describes that numerous disease causing organismscause an increase in nitric oxide production of the body. Evidencesuggests that this production is therapeutic, although too much nitricoxide is also implicated in some disease states.

Circadian variation in nitric oxide has been inversely associated withcircadian variation in blood pressure. Healthy individuals exhibithigher levels of NO as well as higher circadian variation thanindividuals with essential hypertension or peripheral arterial occlusivedisease. See “Role of Endogenous Nitric Oxide in Circadian BloodPressure Regulation in Healthy Humans and in Patients with HypertensionAtherosclerosis” Bode-Boger S M, et al. (J Investig Med 2000 March;48(2)125-32). The effects of nitric oxide on the vascular system aredescribed in “Nitric Oxide and the Regulation of the PeripheralCirculation”, Phillip J. Kadowitz and Dennis B. McNamara, editors,Birkhauser Boston, 2000, as well as in “The Haemodynamic Effects ofNitric Oxide”, edited by Robert T. Mathie and Tudor M. Griffith,Imperial College Press, 1999.

Control and regulation of the generation and release of nitric oxide mayprovide a method to maintain proper blood pressure, vascular tone,coagulation properties of the blood and a host of other bodilyfunctions. However, nitric oxide has a short lifetime in physiologicalfluids.

Hemoglobin can reversibly bind nitric oxide to form S-nitrosohemoglobin.This compound forms in one part of the body and is transported by theblood to regions of reduced oxygen partial pressure where it decomposesreleasing nitric oxide. The nitric oxide then causes dilatation of thecapillaries where the oxygen content of the blood is low. Thisdilatation increases blood flow to those areas where it is needed most,those areas with reduced oxygen. A known source of S-nitrosohemoglobinis the lungs. Nitric oxide is produced in the nasal passages and isabsorbed in the lungs improving the function of the lung by improvingthe match of blood and air flow. The nitric oxide also has effects onperipheral circulation.

It is known that nitric oxide gas may be administered, and is alsogenerated in nasal passages during inhalation, which is drawn into thelung along with inhaled air. Thus nitric oxide is absorbed in the lungwhere it attaches to hemoglobin and forms S-nitrosolated hemoglobin.This is a major source of S-nitrosolated hemoglobin producing systemiceffects in the body. The following U.S. patents disclose variousphysiological effects of nitric oxide inhalation: U.S. Pat. No.5,427,797 issued to Frostell, et al. Jun. 27, 1995; U.S. Pat. No.5,765,548 issued to Perry Jun. 16, 1998; and U.S. Pat. No. 5,904,938issued to Zapol, et al. May 18, 1999.

Topical application of nitric oxide is known. U.S. Pat. No. 5,519,020issued to Smith, et al. May 21, 1996, discloses the use of nitric oxidereleasing materials, placed in close proximity to wounds to enhancehealing through a variety of mechanisms. A polymeric material is used tocontrol the rate at which nitric oxide is released because nitric oxidemay be toxic and injurious in excessive doses.

U.S. Pat. No. 5,646,181 issued to Fung, et al. Jul. 8, 1997 disclosestopical medications containing organic nitric oxide releasing compoundsthat when topically applied release nitric oxide in sufficientquantities to treat impotence without producing systemic side effectssuch as hypotension.

U.S. Pat. No. 5,648,101 issued to Tawashi Jul. 15, 1997 disclosesproducts that liberate nitric oxide through reaction of an inorganicnitrite and a ferrous metal salt. These products may be ingested,applied topically, taken as suppositories, applied as transdermalpatches, and used in osmotic pumps.

U.S. Pat. No. 5,891,472 issued to Russell Apr. 6, 1999 discloses the useof topically applied nitric oxide donors for the treatment of Equinelaminitis.

U.S. Pat. No. 5,895,658 issued to Fossel April 20, 1999, discloses theuse of topically applied L-arginine, a substrate for production ofnitric oxide form nitric oxide synthase to cause local vasodilatation ofthe skin for the purpose of producing beneficial effects such as warmingof cold or cool tissues, growth of hair on the scalp, healing of legulcers secondary to diabetes or confinement to bed, as well asbeneficial effects through restoration of natural mechanisms based onimprovement of local blood supply.

U.S. Pat. No. 5,958,427 issued to Salzman, et al. Sep. 28, 1999,discloses compounds containing nitric oxide donors that do not passthrough mucosal membranes but release nitric oxide which does crossthrough skin or mucosal membranes and provide local effects. Thesecompounds may also be applied topically to the genitalia for used as anaphrodisiac or applied in the mouth for use as antibacterial agents.

U.S. Pat. No. 6,056,966 issued to Selim, et al. May 2, 2000 disclosesthe use of topical organic nitrates which are nitric oxide donors in thetreatment of male impotence. These produce the desired effects withoutundesired systemic effects including hypotension.

Compounds that release nitric oxide may also be injected into the bodyof a subject. U.S. Pat. No. 5,721,278 issued to Garfield, et al. Feb.24, 1998, discloses the use of inhibitors of nitric oxide synthesis toinhibit ovulation, and the use of nitric oxide precursors to bring aboutovulation.

U.S. Pat. No. 5,800,385 issued to Demopulos, et al. Sep. 1, 1998,discloses solutions including nitric oxide donors for irrigating thesites of operative wounds. The nitric oxide donors may be included inthe solutions for their anti-spasm activity.

U.S. Pat. No. 5,858,017 issued to Demopulos, et al. Jan. 12, 1999,discloses the use of solutions containing among other things, nitricoxide donors in urological irrigation solutions.

U.S. Pat. No. 5,861,168 issued to Cooke, et al. Jan. 19, 1999 disclosesthe intramural application of nitric oxide precursors during coronaryballoon angioplasty to reduce thickening of the treated vessels and toimprove tolerance to the angioplasty procedure.

It is known to use nitric oxide to treat a variety of conditions. U.S.Pat. No. 5,278,192 issued to Fung, et al. Jan. 11, 1994, discloses usingorganic nitrates for continuous treatment of conditions including,angina, particularly chronic, stable angina pectoris; ischemic diseases,congestive heart failure, for controlling hypertension and/or impotencein male patients. These organic nitrates may be administered in avariety of ways including sublingual, oral and buccal tablets as well ascapsules, topical creams and ointments, patches, tapes, spray andintravenous solutions.

U.S. Pat. No. 5,385,940 issued to Moskowitz Jan. 31, 1995 discloses theadministering nitric oxide donors or L-arginine to act as the substrateof nitric oxide synthase during a stroke to increase nitric oxideproduction and so cause vasodilatation to reduce the infarct size.

U.S. Pat. No. 5,632,981 issued to Saavedra, et al. May 27, 1997,discloses polymers containing bound nitric oxide releasing compounds.

U.S. Pat. No. 5,645,839 issued to Chobanian, et al. Jul. 8, 1997disclose the use of nitric oxide donors and precursors combined withangiotensin converting enzyme inhibitors to suppress and reversefibrosis in the body, wherein the fibrosis is associated with a disorderselected from the group consisting of cardiovascular fibrosis,arterioscleriotic disorders, pulmonary fibrosis, adult respiratorydistress syndrome, inflammatory disorders, scleroderma, cirrhosis,keloids, and hypertrophic scars.

U.S. Pat. No. 5,648,393 Stamler, et al. July 15, 1997 disclose the useof S-nitrosylated compounds to treat impotence.

U.S. Pat. No. 5,650,447 Keefer, et al. Jul. 22, 1997 discloses the useof polymers containing bound nitric oxide releasing compound to treatrestenosis when incorporated into devices such as sutures, vascularimplants, stents, heart valves, drug pumps, drug-delivery catheters,self-adhering means such as endoluminal implants, liposomes,microparticles, microspheres, beads, disks or other devices.

U.S. Pat. No. 5,789,447 issued to Wink, Jr., et al. Aug. 4, 1998,discloses a method of reducing free radical induced tissue damageassociated with ischemia reperfusion injury wherein the ischemiareperfusion injury is associated with a condition or disease selectedfrom the group consisting of transplantation, trauma, inflammation,stroke, seizure, rheumatoid arthritis, atherosclerosis, cancer,dementia, diabetes, hypertensive crisis, ulcers, lupus, sickle cellanemia, ischemic bowel syndrome, pulmonary emboli, Ball's syndrome,pancreatitis, heart attack, and aging.

U.S. Pat. No. 5,814,666 issued to Green, et al. Sep. 29, 1998, disclosethe use of nitric oxide releasing compounds as antimicrobial agents.

U.S. Pat. No. 5,910,316 issued to Keefer, et al. Jun. 8, 1999 disclosesthe use of a nitric oxide releasing agent directly applied to the penisby delivery means being selected from the group consisting of atransurethral applicator, a penile implant, a dermal patch and a condom.

U.S. Pat. No. 6,057,367 issued to Stamler, et al. May 2, 2000 discloseusing a variety of methods to manipulate nitrosative stress. Thesemethods include using acidified nitrite as a mouth rinse and a mixtureof acidified nitrite plus a thiol as a topical application.S-nitrosothiol may be applied topically or formed an situ from aninorganic nitrite, a pharmacologially acceptable acid and a thio.Pathenogenic microbes may also convert substrates to nitrosating agentswhich inhibit the growth of the pathenogenic microbe.

In the book, “Nitric Oxide and Infection”, Ferric C. Fang ed., KluwerAcademic/Plenum Publishers, 1999, in a chapter titled “Nitric Oxide andEpithelial Host Defense” by Nigel Benjamin and Roelf Dykhuizen, theauthors discusses their findings of nitric oxide production on the skin,the importance of this in normal infection control, and their findingthat a salve containing acidified nitrite is effective in the treatmentof tinea pedis (athlete's foot). They attribute the normal production ofnitrite on the skin to the reduction of sweat nitrate to nitrite by skinbacteria Many heterotrophic bacteria will reduce nitrate to nitrite, forexample, E. coli. These bacteria are facultative anaerobes that normallyutilize oxygen as the electron sink for their cellular respiration, butcan also utilize nitrate in the absence of oxygen. All these bacteriautilize organic substrates for energy, and growth and many of thesebacteria can be pathogenic. In the mouth, salivary nitrate is reduced bythese facultative anaerobes. These nitrate reducing bacteria are keptanaerobic by the layers of biofilm that accumulates on the tongue. Inthat the surface of the skin is expected to be aerobic, reduction ofnitrate to nitrite should be minor. While some nitric oxide may beproduced by bacterial reduction of sweat, the urea content of sweat ismuch higher than that of nitrate. I have found a more significant sourcethat is more easily and safely stimulated is the utilization of urea insweat to form nitrite through ammonia and urea oxidizing bacteria.

Nitrate in the diet is rapidly absorbed and is concentrated by the bodyin the saliva. In the mouth facultatively anaerobic bacteria on thetongue metabolize nitrate to form nitrite. Saliva contains significantnitrite and studies have shown that when the skin is licked that NO isreleased. This NO is believed to have anti-microbial and vasodilatoreffects. The release of NO is the rationalization as to why animals (andhumans) lick wounds to enhance healing. Similarly a common folk remedyfor impotence is the use of saliva directly applied to the penis whereNO release would induce and prolong erection.

In their chapter, Benjamin and Dykhuizen, in discussing the role thatsalivary nitrite has in reducing food born illness, point out that inthe stomach, chloride is present in a high concentration and willcatalyze nitrosation reactions to form additional reactive intermediatesthat may add to the toxicity of acidified nitrite. On the skin, theconcentration of chloride can reach that of a saturated salt solution,levels much higher than can be reached in the stomach.

Bathing has as one of its primary objectives removing bacteria from theskin. While pathogenic bacteria are undesirable, all bacteria are notPathogenic. Recent advances in soap formulations have included theadding of broad-spectrum anti-microbial agents to soap. Bathing hasgreatly reduced the incidence of water-borne diseases such as choleraand various diarrhea diseases. It may be that removal of all bacteriahas the undesired effect of removing the natural bacteria that producenitrite, which the body has evolved to utilize physiologically.

With this in mind a number of curious aspects of human physiology can beunderstood. The areas of the body that are most in need of rapidhealing, infection control and hence of NO production are feet, hands,scalp, and genital area, which are the parts of the body whererespiration is most abundant even when not needed for cooling. This maybe why there is urea, chloride, and iron in perspiration, and whyperspiration has a low pH.

Sauna and other types of sweat baths can be seen as was of enhancing theproduction of nitrite and NO on the skin. Modern use of the sauna aspart of a bathing ritual involving washing with soap and running waterwould not achieve such a result and has only been practiced since the19^(th) century. Urea and nitrite are very water soluble and would bewashed off readily. When the custom of, sauna first developed over 2000years ago, there was no running hot water, so the skin would retain thesoluble urea and nitrite, and without soap the bacteria would also beretained.

Another custom, the use of a whisk, a bundle of birch branches used togently beat the skin, can be seen as a method of inoculating the skinWith bacteria present on the whisk. Between uses the whisk would dry outand the bacteria surviving would necessarily become adapted to living onperspiration residues finder fairly dry conditions, the natural state ofhuman skin. The advent of the germ theory, and the perceived need foraseptic hygienic conditions has modified the use of such devices towhere they probably no longer serve this original function.

The beneficial health effects of sweating can be seen a deriving fromthe increased production and release of NO on the skin, rather than dueto removal of wastes. Perspiration as a waste removal method would seemto be a non-intuitive and ineffective method. Most sweating occursduring periods of high metabolic load, which would seem to be aninopportune time to use any metabolic capacity to rid the body of wasteproducts. In fact the kidneys shut down under conditions of insufficientheart output. Sweat output can vary greatly from day to day, hour tohour, and minute to minute. Accumulating wastes in anticipation ofepisodic sweating events would seem to be a poor allocation ofResources. Nervous sweating that occurs in anticipation of stressfulevents may be the body's way or preparing itself for a stressful event.Dampening the skin with perspiration would release NO from the newlygenerated and accumulated nitrite that would ten act as a vasodilator,which may enhance blood flow and prepare the body to respond effectivelyto the stressor. Organic nitrates like nitroglycerine are oftenprescribed for exactly such use prior to physical or emotional stress toachieve just such vasodilatation.

Sweating can then be seen as the solution to skin disorders, rather thanas the cause. If the ammonia and urea oxidizing bacteria content of theskin were restored to pre-industrial levels, then areas of the body withprofuse sweating would also have profuse nitrite and NO production andwould be expected to heal faster, better resist infection, and be in abetter general state of health. In the absence of such bacteria, whichhappen to be relatively slow growing, other faster growing heterotrophicbacteria would hydrolyze urea to free ammonia which is quite toxic andirritating to the skin. It is thus the absence of the proper bacteriathat cause perspiration residues to become irritating. Oxidation ofammonia to nitrite or nitrate lowers the pH, and converts any remainingfree ammonia to the less toxic ammonium ion.

After formation on the skin, nitric oxide may diffuse into thecapillaries of the skin and be taken up by the blood. The capillaries ofthe skin may dilate in response, and some of the nitric oxide may betaken up by hemoglobin to form S-nitrosohemoglobin which will circulatethough out the body and have systemic effects.

A region of the skin that is thin and has abundant blood capillaries isthe skin of the head and scalp. The presence of hair on the head isoften rationalized as limiting heat loss. But one wonders why the scalphas hair to prevent heat loss while the rest of the body remainsessentially hairless. There is significant heat loss from the head,possibly a result of the skin of the scalp being thin so as to allow therapid diffusion of nitric oxide into the blood. The blood from the scalpjoins that from the brain before entering the heart and lungs. The bloodsupply of the brain is among the most critical to the body and showslittle variation even during periods of extreme metabolic stress.Combining blood having low oxygen tension from the brain with bloodhaving a high nitric oxide content from the scalp may be an efficientuse of the nitric oxide so produced. Because nitric oxide may also bereleased into the air around the head and face, where air drawn induring breathing in close proximity to a source of nitric oxide, theconcentration of nitric oxide would increase in inspired air. Thedifferent patterns of facial hair seen between men and women may derivefrom different patterns of peak metabolic activity, men during huntingand fighting and women during pregnancy, labor and childbirth. Just asinhaled nitric oxide is protective of pulmonary hemorrhage for horses,nitric-oxide released by bacteria on facial hair may facilitate greaterlevels of physical exertion.

Many of the veins of the scalp drain through the skull into the vascularsinuses in the brain. The arteries bringing blood to the brain passthrough these same sinuses. This contact may be so that nitric oxide maydiffuse from the venous blood leaving the scalp into the arterial bloodentering the brain. Such diffusion would help explain the protectiveeffect of moderate exercise on stroke. Sweat on a head would then reducethe vascular resistance in the brain.

Hair can be seen as an insulating material but also as a surface onwhich ammonia oxidizing bacteria may proliferate. Hair may also be anabsorbent material to prevent sweat from dripping off and may alsoprovide a suitable micro-climate for the ammonia oxidizing bacteria.Nervous sweat is mediated through the adrenergic pathway and typicallystimulates sweat on the head and neck.

Sweating for non-cooling purposes can be seen as a natural way for thebody to increase nitric oxide production. The malaria causing organismis affected by nitric oxide, and when nitric oxide synthase inhibitorsare given to animals with malaria, the mortality is increased. Theexcessive sweating that is one of the symptoms of malaria (and of manyother infections) can be seen as one of the body's natural nitric oxideincreasing mechanism.

Human sweat has a high concentration of lactic acid. This renders thenormal pH of sweat in the 4.5 range, where nitrite rapidly decomposes torelease NO. Sweat also contains abundant iron. A quarter of ingestediron is excreted in the sweat. Iron is well known to coordinate with NOand form iron-nitrosyl complexes. Nitric oxide also reacts withsuperoxide to form peroxynitrite. In aqueous solution iron catalyzesreactions of peroxynitrite with other compounds to form toxic products.The presence of abundant iron and nitrite on the skin may be the firstline of defense against skin infections.

Because the outer layers of skin are non-living, pickling and curing ofthese dead layers with nitric oxide prior to their sloughing off shouldhave no detrimental health effects. Indeed, depending on bacteria fornitrite production and hence NO production, no living part of the bodyneed be exposed to high levels of nitrite or nitric oxide or toxic NOreaction products. Levels can be reached on the skin what would bedetrimental to living tissues. This may be a very effective way ofwarding off skin infections, and may be the system that humans haveevolved to utilize in the absence of frequent bathing.

The effect of exercise on vascular disease can also be seen in a newlight. Moderate activity is both exercise and a way of, through inducingperspiration, increasing NO production. Vigorous activity does thesethings also, but if the sweating is sufficiently profuse, bathing isgenerally done after the exercise. Washing away the perspiration removesthe protective effect of this NO production. This explains the reducedprotective effect of vigorous exercise when compared with moderateexercise seen in some studies. The protective effect of exercise isincreased but the protective effect of NO is reduced by bathing. Thatmay indicate that the beneficial health effect of skin bacteria derivedNO is, at least in some people, of comparable magnitude to that ofexercise.

Reduced incidence of heart disease in summer may be due to the increasedamount and time that sweat stays on the skin. In that the ammoniaoxidizing bacteria are slow growing, they can easily be washed offfaster than they can proliferate. That an annual pattern can be seen inthe incidence of heart disease is strong evidence that this effect issubstantial.

Nitrite can be generated in several ways by bacteria. The first is bythe oxidation of ammonia or urea. This is the necessary first step inthe nitrification of ammonia in soil. Specific autotrophic bacteriautilize this ammonia oxidation to provide all their metabolic energyneeds. A second type of autotrophic bacteria utilize this nitrite andfurther oxidize it to nitrate and utilize this energy for theirmetabolism. Other bacteria including heterotrophic bacteria can utilizenitrate to oxidize other compounds while reducing the nitrate tonitrite. At or below pH 5.5 nitrite decomposes releasing NO.

The reduced incidence of heart disease may be due to nitric oxidederived from the bacterial reduction of nitrate or to the bacterialgeneration nitrite from ammonia or urea. Autotrophic bacteria are onlyexpected to proliferate if there is a long interval between bathing,weeks or more, so that much of the observed differences in incidence islikely due to nitric oxide derived from sweat nitrate. However the useof ammonia oxidizing bacteria in the present invention would allow forhigher levels and larger effects on vascular health.

Similar processes are used to generate nitric oxide that is used incuring meat. General properties of nitric oxide, physiologicalproperties, chemical properties, and its role and mechanism of action infood preservation is well described in a book “Nitric Oxide Principalsand Actions”, edited by Jack Landcaster, Jr., Academic Press, 1996.Heterotrophic bacteria, to achieve a low pH and to produce nit-te fromnitrate, are commonly used in meat preserving where nitrate in apickling brine is reduced to nitrite, releasing NO which reacts withmeat to produce the characteristic color and flavor of cured meat. Inmeat preserving, these bacteria are added as a pure culture where theyretard the undesirable growth of disease and spoilage bacteriaMicrococcus varians is sometimes used for this purpose as described inU.S. Pat. No. 4,147,807 issued to Gryczka, et al. Such bacteria, as anormal component of food, would not be expected to cause any adversehealth effects when applied to the skin or when accidentally ingested.Applying such bacteria to the skin would enhance the production ofnitric oxide on the skin through the reduction of nitrate to nitrite.Some heterotrophic bacteria can produce nitrite from ammonia, but theiroxidation of ammonia is substantially slower than that of theautotrophic lithotrophic ammonia oxidizers.

In the environment, ammonia and urea are oxidized to nitrite byNitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus,and Nitrosovibrio. These bacteria are all lithotrophic Gram-negativebacteria that utilize carbon dioxide as their major carbon source. Inthe environment nitrite is oxidized to nitrate by Nitrobacter andNitrocystis. Nitrosomonas is the most abundant of these types in soiland would be expected to be the most abundant on normal skin. Thesebacteria are autographs, that is, they do not utilize organic carbon forenergy although some can assimilate organic carbon to a limited extentwhich can stimulate growth. All metabolic energy is obtained from theoxidation of the nitrogen containing species. The majority of carbonderives from the fixing of carbon dioxide utilizing this energy. Becausethese bacteria need only ammonia, oxygen inorganic minerals, and carbondioxide, they are expected to be completely non-pathogenic. The onlypart of the body where all of these are available is the exterior of theskin. They are slow growing when compared to other bacteria Where E.coli has an optimum doubling time of 20 minutes, Nitrosomonas has anoptimum doubling time of 10 hours. Because they do not utilize glucoseor other organic compounds, they are difficult to culture and do notgrow on the standard media used for isolating pathogens, which doutilize organic substrates for energy and growth. Some strains can alsoutilize urea directly.

These autotrophic ammonia oxidizing bacteria can be stimulated byapplying to the skin nutrients needed by these bacteria such as thosenutrients found in American Type Culture Collection standard culturemedia, including ATCC 1953, ATCC 928, ATCC 1573, ATCC 221, ATCC 929,including, for example, urea, ammonium salts, sodium, potassium,magnesium, calcium, phosphate, chloride, sulfate, trace mineral saltsincluding iron, copper, zinc, cobalt, manganese, molybdenum and buffers.Application of a preparation or solution comprising some or all of thosenutrients to the skin and scalp would stimulate the naturally occurringautotrophic bacteria, forming nitrite and nitric oxide withoutstimulating heterotrophic bacteria.

Nitrobacter are inhibited by elevated pH and by free ammonia In soilthis can lead to the accumulation of nitrite in soil which is quitetoxic when compared to nitrate. On the skin, addition of an alkalineagent would raise the pH and inhibit the oxidation of nitrite allowinghigher concentrations to develop. Thus using an alkaline compound couldserve to increase the concentration of nitrite. Talc while beingessentially neutral often contains calcium and magnesium carbonates asimpurities. Small amounts of these may then make the skin alkaline whendry, but upon sweating the pH would drop and the increased nitrite wouldbe available for conversion to NO. Inhibiting bacteria such asNitrobacter that reduce the nitrite concentration on the skin is auseful method to further enhance nitric oxide release. Alternatively,Nitrobacter may be included, which will then increase the production ofnitrate. Then other bacteria utilizing this nitrate and the otherorganic compounds on human skin to form nitrite can be used.

Bacteria that are useful in this regard are bacteria that metabolize thenormal constituents of human perspiration into NO precursors. Theseinclude, for example, urea to nitrite, urea to nitrate, nitrate tonitrite, urea to ammonia, nitrite to nitrate, and ammonia to nitrite. Insome cases a mixed culture is preferred. The bacteria can convenientlybe applied during or after bathing and can be incorporated into varioussoaps, topical powders, creams, aerosols, gels and salves. One aspect ofthe invention contemplates application to body parts that perspire themost, such as, for example, hands, feet, genital area, underarm area,neck and scalp. The major difference between these different areas ofthe skin is the activity of water. The of the hands is much drier thanthat of the feet, normally covered with socks and shoes, due to theincreased exposure of the hands to the drying effects of ambient air. Itis contemplated that different strains of bacteria may work best ondifferent areas of the body, and a mixed culture of all the types wouldallow those that grow best to proliferate and acclimate and become thedominant culture present in a specific area. Clothing may also be wornto change the local microclimate to facilitate the growth of the desiredbacteria. For example, wearing a hat may simulate dense hair and help tomaintain the scalp in a warmer and moister environment.

Because a normal skin environment is relatively dry, bacteria adapted tolow water tension environments are advantageous. One example of amoderately halophilic ammonia oxidizing bacteria is Nitrosococcusmobillis described by Hans-Peter Koops, et al. (Arch. Microbiol. 107,277-282(1976)). This bacteria has a broad range of growth. For example,while the optimum pH for growth is 7.5, at pH 6.5 it still grows at 33%of its maximal rate. Another more halophilic species, Nitrosococcushalophillus described by H. P. Koops, et al. (arch. Micorbiol. (1990)154:244-248) was isolated from saturated salt solutions in a naturalsalt lake. Nitrosococcus oceanus (ATCC 1907) is halophilic but has anoptimum salt concentration intermediate between the other two. Theoptimum NaCl concentrations for the three are 200, 700, and 500 mM NaClrespectively. N. oceanus however utilizes urea and tolerates ammoniaconcentrations as high as 1100 mM as ammonium chloride. While growth atoptimum conditions is the fastest, similar results may be achieved byusing more bacteria. Thus while th optimum pH for growth of N. mobillisis 7.5, one can achieve the same nitrite production by using 3 times asmany bacteria at pH 6.5. Because the quantities of bacteria in thepresent invention may be large, a number of orders of magnitude largerthan that which occurs within 24 hours of bathing, the fact that the pHof the skin is not optimum for these bacteria is not an inhibition totheir use. Because N. halophillus was isolated from a saturated saltsolution, it should easily survive the relatively moister human skinenvironment.

Some bacteria produce nitric oxide directly. One example is described in“Production of nitric oxide in Nitrosomonas europaea by reduction ofnitrite”, by Armin Remde, et al. (Arch. Microbiol. (1990) 154:187-191).N. europaea as well as Nitrosovibrio were demonstrated to produce nitricoxide directly. Nitrosovibrio is often found growing on rock where theacid generated causes corrosion. It has been suggested by Poth and Foch,“Dinitrogen production from nitrite by a Nitrosomonas isolate.” (ApplEnviron Microbiol 52:957-959), that this reduction of nitrite tovolatile nitric oxide is used as a method for the organism to eliminatethe toxic nitrite from the environment where the organism is growing,such as the surface of a rock.

Any ammonia oxidizing bacteria may be used in the present invention. Ina preferred embodiment, the ammonia oxidizing bacteria may have thefollowing characteristics as are readily known in the art: ability torapidly metabolize ammonia and urea to nitrite and other NO precursors;non pathogenic; non allergenic; non producer of odoriferous compounds;non producer of malodorous compounds; ability to survive and grow inhuman sweat; ability to survive and grow under conditions of high saltconcentration; and ability to survive and grow under conditions of lowwater activity.

Natural bacteria can be used as well as bacteria whose characteristicshave been altered through genetic engineering techniques. Bacteriaculturing techniques can be used to isolate strains with the abovecharacteristics. A mixture of pure strains would avoid the problemsassociated with simply culturing bacteria from the skin, which includesthe potential growth of pathogens and other bacteria having undesirablecharacteristics. However, culturing bacteria from the skin and growingthem on growth media that simulates the composition of humanperspiration may also be effective at increasing the nitric oxideproduction rate. A useful method for culturing and isolating suchbacteria is to grow them on media containing urea and ammonia plusmineral salts, but without the organic compounds that heterotrophicbacteria utilize, such as sugars and proteins. When isolatingautotrophic ammonia and ammonia oxidizing bacteria, it may also bedesirable to attempt growth on a heterotrophic media to verify that theautotrophic strain is not contaminated with heterotrophic bacteria.

U.S. Pat. No. 4,720,344 issued to Ganczarczyk, et al. Jan. 19, 1988,discloses the operation of a waste water treatment facility underconditions that maximize the conversion of ammonia to nitrite whileminimizing the conversion of the resulting nitrite to nitrate. This isaccomplished by utilizing conditions that are conducive to the growth ofNitrosomonas but not to Nitrobacter. This is accomplished mostpreferably by adjusting the pH and ammonia content of the waste water tolevels that are conducive to the growth of Nitrosomonas but not toNitrobacter and then adjusting the hydraulic retention times in thecontacting chambers to less than the recovery time of the inhibitedbacteria.

U.S. Pat. No. 5,314,542 issued to Cassidy, et al. May 24, 1994,discloses the growth and treatment of bacterial cultures of Nitrosomonasto allow for extended shelf life in a dormant state and subsequenttreatment to produce rapid recovery of metabolic activity.

U.S. Pat. No. 5,139,792 issued to Ware, et al. Aug. 18, 1992, disclosesa method of dispensing specific bacteria into animal Fed and drinkingwater without agitation to maintain the bacteria in suspension.

An analogous method of treatment of ammonia oxidizing bacteria culturesis also useful in the present invention where ammonia oxidizing bacteriaare grown in a media, concentrated and separated from the media,suspended in sterile water with the proper salt concentration, storedunder aseptic conditions, reviving the bacteria through addition ofammonia, and then applied to the skin.

Methods of isolation of useful bacteria suitable for colonization ofhuman skin are analogous to methods used for the isolation of bacteriasuitable for colonization of livestock digestive systems. Scrapings arecollected from healthy individuals, inoculated into suitable media,grown and characterized. Steady state continuous culture methods can beused to ensure stability of the culture over time.

A useful method of treatment of ammonia oxidizing bacteria cultures,along the lines of Cassidy, et al. in U.S. Pat. No. 5,314,542, is whereammonia oxidizing bacteria are grown in a media, concentrated andseparated from that media, suspended in sterile water with the propersalt concentration, stored under septic conditions, revived throughaddition of ammonia, and held for a period of time for the bacteria tobecome active. Cassidy, et al. utilize their bacteria culture andstorage method for bacteria used for ammonia control in aquaria. Asimilar method of growing and treating bacteria can be used to producebacteria which can then be used in the present invention and applied tothe skin.

The ammonia oxidizing bacteria are aerobes which requires oxygen fortheir metabolism and cannot grow in anaerobic conditions. However manyof them can also use nitrate as well as oxygen as the terminal electronsink of their metabolic processes. Storage for prolonged periods of timein a sealed container runs the risk of the container becoming anoxic.Nitrate can be added to the fluid in the container so that nitrate canbe utilized instead of oxygen for bacteria respiration during storageallowing for non-fluid formulations such as gels and sticks. Bacteria onthe interior of such formulations can derive their oxidizing substratefrom dissolved nitrate in the absence of dissolved oxygen.

In another embodiment of the present invention, urea, nitrite andnitrate, iron, lactic acid, and sale may be included in a compoundcomprising the bacteria or applied separately to supplement the skin,because bathing removes these water soluble compounds. The bacteria mayalso be applied during or after bathing and may be incorporated intovarious topical powders, creams, sticks, aerosols, and salves. Othercompounds may be added to these cosmetic preparations as selected by oneskilled in the art of cosmetic formulation such as, for example, water,mineral oil, coloring agent, perfume, aloe, glycerin, sodium chloride,sodium bicarbonate, pH buffers, UV blocking agents, silicone oil,natural oils, vitamin E, herbal concentrates, lactic acid, citric acid,talc, clay, calcium carbonate, magnesium carbonate, zinc oxide, starch,urea, and erythorbic acid.

The ammonia oxidizing bacteria, may be applied to any surface of asubject, such as, for example, skin and hair. In a preferred embodiment,the bacteria is applied to the skin of a subject. In a more preferredembodiment, the ammonia oxidizing bacteria may be applied to the scalpbecause the scalp provides excellent blood supply. Bacteria may beincorporated into various hair treatments and devices, includingconditioners, gels, hair sprays, hair nets, combs, brushes, hats, hairpieces.

Another embodiment of the invention includes analogous methods used forcuring meat, since the goal of meat curing is the production of nitricoxide. These pickling brines and curing compositions are expected topresent little health risk since they are considered safe for humanconsumption. In particular, when nitrite is treated with ascorbic acid,nitric oxide is produced. Nitrite is reduced by ascorbate to generatenitric oxide. Usually in modern meat curing, ascorbate or erythorbateare used with nitrite to generate nitric oxide. In meat preserving,nitric oxide is a precursor in a chain of chemical reactions leading tothe development of specific flavors and colors. At low pH, such as lessthan 5, nitric oxide is rapidly lost from pickling brines before thesechemical reactions can occur. Therefore, a higher pH is recommended formeat preserving. Mixtures of nitrite with erythorbate may be one suchexample. Combining meat curing formulations with cosmetic typeformulations may achieve a similar benefit. Combining bacteria, urea,and erythorbic acid may be a preferred combination. Otherphysiologically acceptable acids may be used as well.

An advantage of an embodiment of the invention is that the induced NOproduction is under physiological control through sweating. Organicnitrates, such as nitroglycerine, are sometimes prescribed for use priorto time c f emotional or physical-stress. These are the same conditionsunder which nervous sweating occurs. One aspect of the present inventionmay be a reduced incidence of heart disease, vascular diseases,impotence, and infertility. Any condition that may be treated through aNO enhancing method may be amenable to treatment with present invention,Even where known treatments include administering nitric oxide or NOdonor substances orally, topically, sublingually, nasally, by injection,by inhalation. For example, impotence is treated with Viagra, whichextends the duration of action of NO. Use of th present invention mayreduce the need for the use of agents such as Viagra.

Individuals need not have clinical symptoms of any of these disorders inorder to benefit from the present invention. The invention may be usedas a preventative measure along the same lines as proper diet, takingvitamins, exercising, or as bathing in general. Because disorders arerelated through the common action of the vasodilator NO, one, can usethe invention for heart disease prevention and receive a therapeuticvalue for impotence. Because impotence is a disorder that is oftenstigmatized, an impotence treatment that can be disguised as a generalhealth tonic is advantageous.

In another embodiment of the invention, the bacteria is applied to thesurface of non-human vertebrates. Domesticated animals such as horses,dogs, pigs, and chickens are seen to roll in and cover themselves withdirt. In that urea is an abundant compound in urine and manure, bacteriaadapted to living in barnyard soil would be expected to be rapidmetabolizers of urea into nitrite. Wild animals also cover themselveswith dirt. A component of such behavior is likely the inoculation of theskin or far with bacteria that will metabolize sweat components into NOand NO precursors. Using a substantially pure culture of such bacteriawould improve the health of domesticated animals and facilitate theirgrowth. Ammonia is often present in large amounts in animal feed lotareas. Bacteria that would metabolize ammonia into NO or NO precursorswould reduce the ill effects of ambient ammonia and improve theeconomics of intensive animal farming. Other subjects are for example,vertebrates such as, domesticated, laboratory, transgenic, chimeric, andzoo animals such as, horse, pig, cow, dog, cat, goat, sheep, buffalo,donkey, mule, elephant, cat, wolf, camel, llama, chicken, turkey,primates, ungulates, rodents chimpanzees, gorilla, orangutan, mice,rats, and rabbits.

The practice of some animals, to deposit their urine and feces in asingle location can be seen as thee instinctive production of a richenvironment for the culturing and proliferation of nitrite producingbacteria. That animals instinctively exhibit behaviors that re-inoculatetheir skins with these bacteria would indicate that these bacteria canbe readily lost from the skin and that re-inoculation is necessary foranimals. In that humans typically bathe more frequently than animals,the human need for re-inoculation is correspondingly greater.

Treatments for foundering or equine laminitis as described in U.S. Pat.No. 6,045,827 and 5,891,472 issued to Russell; Meri Charmyne both titled“Treatment of equine laminitis” include the treatment of this conditionwith nitric oxide donors applied topically to the affected areas. Thisserious disease of horses is treated through application of a nitricoxide donor to the feet and hoof region. Nitroglycerine has been used,as have other nitric oxide donors. Horses instinctively accomplish thisin the wild by urinating in the mud, allowing nitrite forming bacteriato proliferate, and walking through this mud containing the nitriteproducing active cultures. Modern stable practices call for good housekeeping and the elimination of any accumulation of urine and feces wherehorses walk. All hoofed animals are subject to similar disorders of thefeet and hooves. Thus all hoofed animals may benefit from application ofthe suitable ammonia oxidizing bacteria.

U.S. Pat. No. 5,765,548 issued to Perry discusses the use of nitricoxide mixed with air breathed by the horse during vigorous exercise andthe use of nitric oxide augmenters injected prior to exercise asbeneficial in reducing the incidence and severity of exercised inducedpulmonary hemorrhaging. Significant reductions in capillary pressurewere demonstrated by Perry. Application of an appropriate ammoniaoxidizing bacteria to the horse's skin may have the effect of increasingthe natural production of nitric oxide during exercise. This nitricoxide may diffuse through the horse's skin and be absorbed into theblood where it would circulate resulting in systemic effects. Somenitric oxide would also be released into the air around the horse andwould be inhaled. Presumably decreased pressure drop translates intoincreased maximal flow of air and blood in the lungs, and henceincreased maximal exercise performance. Achieving this increasedperformance through natural means would be advantageous in horse racing.Similarly racing gray hounds, draft animals, beasts of burden, andanimals under stress may also have their nitric oxide productionenhanced. Human athletes may similarly enhance their performance byutilizing skin bacteria to augment nitric oxide production before,during and after exercise. Typical athletic events include, for examplefoot races, weight lifting, bicycle face or practice, football game orpractice, soccer game or practice, basket ball game of practice,baseball game or practice, golf game or practice, mountain climbing,boxing match or practice, hockey game or practice, and tennis match orpractice.

The ammonia oxidizing bacteria may be positioned in lose proximity to asurface of a subject by being applied directly or indirectly to thesurface of the subject. Suitable bacteria may be positioned in closeproximity to the surface of the subject by being indirectly applied byapplication to articles with which the surface of the subject comes intocontact, such as, for example, bedding products such as straw, woodshavings, pillows, sheets, habitat enclosures, stalls, brushes, combs,and mattresses. Similarly suitable bacteria can be added to litter boxproducts so that when the animal comes into contact with the litter andlitter box, the animal subject will be in close proximity to thebacteria. As an added feature of such litter box products the urea inurine may be oxidized to non-volatile products and the ammonia smell oflitter boxes will be reduced. Rather than give off ammonia, the litterboxes would give off nitric oxide which would enhance the pulmonaryfunction of animals and humans in the vicinity, as well as providesystemic effects.

In one aspect of the invention, an article is treated with ammoniaoxidizing bacteria. For example the article may be coated or impregnatedwith the bacteria. In a preferred embodiment, the article treated withthe bacteria, contacts a surface of a subject, such as, for example,clothing, collar, and saddle.

Articles contacting the surface of a human subject, such as a diaper,may be treated with ammonia oxidizing bacteria. Because diapers aredesigned to hold and contain urine and feces produced by incontinentindividuals, the urea in urine and feces can be hydrolyzed by skin andfecal bacteria to form free ammonia which is irritating and may causediaper rash. Incorporation of bacteria that metabolize urea into nitriteor nitrate may avoid the release of free ammonia and may release nitriteand ultimately NO which may aid in the maintenance of healthy skin forboth children and incontinent adults. The release of nitric oxide indiapers may also have anti-microbial effects on disease causingorganisms present in human feces. This effect may continue even afterdisposable diapers are disposed of as waste and may reduce the incidenceof transmission of disease through contact with soiled disposablediapers. The addition of the ammonia oxidizing bacterial to the diaperis beneficial when realizing that cleaning a soiled infant can removeammonia oxidizing bacteria faster than they can proliferate, leavingonly heterotrophic urea hydrolyzing bacteria on the skin. The epidemicof infant deaths due to Sudden Infant Death Syndrome, or SIDS, in the1980's was approximately coincident with the widespread use ofdisposable diapers. The “back to sleep” program where infants are put tosteep on their backs has greatly reduced the incidence of SIDS. Themechanism of the causal relationship between back sleeping and low SIDSincidence remains elusive, however, it may be due to the increasedcontact of infant skin with urine during sleep occurring while theinfant is lying on its back. Victims of SIDS are often found with sweatsoaked bed clothes which may be due to due to the infant's vain attemptto increase nitric oxide formation during asphyxiation by sweating,rather than due to overheating as is conventionally thought.

Another article of clothing that can be so treated is the tampon. Duringa woman's menstrual period, secretions are generated which under certaincircumstances can support the growth of heterotrophic disease causingbacteria such as those that cause toxic shock. Just as topically appliedacidified nitrite has been shown to be curative for yeast infections ofthe skin, it is expected that vaginal application of these bacteriashould be curative and preventative of vaginal yeast infection. Byrendering the vagina less hospitable to disease causing organisms, theincidence of transmission of sexually transmitted diseases can bereduced.

Vaginal use of these bacteria by a woman may also enhance the sexualperformance of a male partner by providing additional nitric oxide toher partner's sexual organ during sexual intercourse. Just as thenitrite in saliva provides the basis for the folk remedy for impotence,that of applying saliva to the male sexual organ, the stimulatory effectof the application of saliva to the female genitalia may also have itsbasis in the nitrite content of saliva. The present invention byenhancing the production of nitric oxide may provide a similar benefitin enhancing the sexual function of both men and women.

Other articles of clothing such as, for example, shoes, shoe inserts,pajamas, sneakers, belts, hats, shirts, underwear, athletic garments,helmets, towels, gloves, socks, bandages, and the like, may also betreated with ammonia oxidizing bacteria. Bedding, including sheets,pillows, pillow cases, and blankets may also be treated with thebacteria. In one embodiment of the invention, areas of skin that cannotbe washed for a period of time may also be contacted with ammoniaoxidizing bacteria. Specifically, skin enclosed in orthopedic castswhich immobilize injured lift bs during the healing process, and areasin proximity to injuries that must be kept drib for proper healing suchas stitched wounds may benefit from contact with the ammonia oxidizingbacteria.

It is contemplated that articles worn about the head and scalp may betreated with ammonia oxidizing bacteria. Nitric oxide formed on thehair, away from the skin surface, may be captured in a hat, scarf orface mask and directed into inhaled air.

Individuals having a reduced bathing frequency, such as astronauts,submarine crew members, military personnel during a campaign, civilianworkers in remote locations, refugees, bedridden individuals and manyothers may maintain healthier skin by maintaining skin bacteriaaccording to the present invention. Bed sores are a common factorderiving from disturbances to blood flow. It is expected that thepresent invention may augment and normalize inadequate circulationproblems.

Another aspect of the invention includes the use of the bacteria toinhibit the growth of heterotrophic bacteria. Body odor derives, inpart, from bacterial metabolites on the skin. The development of skinodor in a day or so indicates that fast growing heterotrophic bacteriagenerate the odoriferous compounds. Ammonia oxidizing bacteria, byinhibiting the growth of the heterotrophic bacteria, may decrease theodor produced. Thus the present invention may also be used to reducebody odor, and may be used alone or in conjunction with other deodoranttype cosmetic preparations.

In another embodiment, garments such as, for example, condoms, andcodpieces may be treated with the proper bacteria. It is known that themale sexual organ requires nitric oxide for proper-function duringsexual intercourse. The stimulation from nitric oxide generation throughwearing the articles may be beneficial for male subjects contemplatingsexual acts. Similarly, the treatment of fabric coverings of furnitureused for sexual activities would also be advantageous, such as, forexample, sheets, blankets, slip covers, pillow cases.

Ammonia oxidizing bacteria may be located on a surface of the articledirectly contacting the surface of the subject. Alternatively, thebacteria may be exposed to bodily fluids but riot directly in contactthe surface of the subject. In particular, a diaper, tampon, or bandagemay have an inner layer treated with the ammonia oxidizing bacteria, andat least one layer that is permeable to bodily fluids, nitric oxide, andor nitric oxide precursors. These layers need not be permeable tobacteria. Because the ammonia oxidizing bacteria cannot utilizecompounds other than ammonia for energy, they cannot infect a wound.Although they may be allergenic, the inhibition of growth ofheterotrophic bacteria may outweigh the potential for allergy.

It is contemplated that different bacteria will be most suitable fordifferent applications. Thus bacteria adapted for very high levels ofnitrite production may be ideal for use in diapers, animal bedding, andother non-contact applications. High nitrite levels would also be usefulfor protecting skin from infections during extended safaris in tropicalenvironments, for military type applications, or for the enhancement ofperformance of elite athletes, human and non-human vertebrate.

While it is expected that the autotrophic ammonia oxidizing bacteriawill be the most active at producing nitric oxide and nitric oxideprecursors, other bacteria producing lessor amounts may be used as well.These may be desired in some circumstances when, for example, bettercontrol of the nitric oxide production is needed. Other bacteria can beincluded for other purposes, such as, for example, to control the pHthrough production of acid.

Further modification and equivalents herein disclose will occur topersons skilled in the art using no more than routine experimentation,and all such modifications and equivalents are believed to be within thespirit and scope of th invention as defined by the following claims.

1. A method of supplying nitric oxide to a subject comprisingpositioning ammonia oxidizing bacteria in close proximity to thesubject.
 2. The method of claim 1, wherein the act of positioningammonia oxidizing bacteria comprises: applying ammonia oxidizingbacteria to a surface of the subject in an effective amount to cause thebacteria to metabolize any of ammonia, ammonium salts, or urea on thesurface into any of nitric oxide, nitric oxide precursors, orcombinations thereof.
 3. The method of claim 2, wherein the act ofapplying the bacteria comprises applying the bacteria in a suitablecarrier.
 4. The method of claim 2, wherein the act of applying thebacteria comprises applying a bacteria selected from the groupconsisting of any of Nitrosomonas, Nitrosococcus, Nitrosospira,Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof. 5.The method of claim 2, wherein the act of applying the bacteria to asurface comprises applying the bacteria to skin, hair, or a combinationthereof.
 6. The method of claim 2, wherein the act of applying thebacteria comprises applying a substantially pure bacteria.
 7. The methodof claim 2, wherein the act of applying the bacteria comprises: applyingthe bacteria to an article; and contacting the article with the surfaceof the subject.
 8. The method of claim 2, wherein the act of applyingthe bacteria comprises applying the bacteria mixed with an acid.
 9. Themethod of claim 2, wherein the act of applying further comprisesmetabolizing any of urine, feces, blood, wound secretions, menstrualsecretions, vaginal secretions, topically applied mixtures, andcombinations thereof. 10 The method of claim 2, further comprising theact of applying a compound selected from any of a component ofperspiration, urea, nitrite, lactic acid, nitrate, salt, iron salts,ammonium salts, and combinations thereof, to the surface of the subject.11. The method of claim 2, wherein the act of applying the bacteriacomprises applying the bacteria to a subject that is a non-humanvertebrate.
 12. The method of claim 2, further comprising: applying atleast one of urea or metal salts to the surface of the subject in aneffective amount to stimulate the growth of the bacteria.
 13. The methodof claim 5, wherein the act of applying the bacteria to a surface of asubject includes applying the bacteria to the skin, hair, or acombination thereof, of a human.
 14. The method of claim 5, wherein theact of applying the bacteria comprises applying the bacteria to thesubject wherein the subject has a disorder selected from any of avascular disease, impotence, infertility, or combinations thereof. 15.The method of claim 7, wherein the act of contacting the article withthe surface of the subject further comprises contacting the bacteriawith surface of the subject.
 16. The method of claim 7, wherein the actof contacting the article with the surface of the subject furthercomprises contacting a fluid excretion from the surface of the subjectwith the bacteria.
 17. The method of claim 7, wherein the act ofapplying the bacteria comprises applying the bacteria to an article ofclothing.
 18. The method of claim 7, wherein the act of applying thebacteria mixed with an acid comprises applying the bacteria nixed withlactic acid.
 19. The method of claim 10, wherein the acts of applyingthe compound and the bacteria include applying compound and the bacteriain a mixture.
 20. The method of claim 11, wherein the act of applyingthe bacteria to the surface of the non-human vertebrate comprises:applying the bacteria to any of skin, feather, hair, fir, scale orcombinations thereof.
 21. The method of claim 13, wherein the act ofapplying the bacteria comprises applying the bacteria to an underarmsurface.
 22. The method of claim 17, wherein the act of applying thebacteria to the article of clothing comprises the acts of: wearing thearticle and impregnating the article with the bacteria.
 23. The methodof claim 17, wherein the act of applying the bacteria to the article ofclothing comprises the acts of: wearing the article and coating thearticle with the bacteria.
 24. The method of claim 17, wherein act ofapplying the bacteria comprises applying the bacteria to a diaper. 25.The method of claim 17, wherein the act of applying the bacteriacomprises applying the bacteria to an article of clothing selected fromthe any of shoe, sneaker, belt, hat, undergarment; pajama, athleticgarment, sock, shoe insert, bandage, face mask, scarf, tampon, andcondom.
 26. The method of claim 20, wherein the act of applying thebacteria comprises applying the bacteria any of bedding, litter, anarticle of clothing, or a habitat.
 27. The method of claim 22, whereinthe act of impregnating the article includes contacting the article witha culture media containing the bacteria.
 28. The method of claim 27,wherein the act of contacting the article with a culture mediacontaining the bacteria comprises applying an animal manure.
 29. Themethod of claim 27, wherein the act of contacting the article with aculture of media containing the bacteria comprises contacting thearticle with a substantially pure culture of bacteria.
 30. The method ofclaim 2, wherein the act of positioning the bacteria comprises: applyingthe ammonia oxidizing bacteria the a surface Of the subject in an amounteffective to metabolize a normal bodily secretion on the surface intoany of nitric oxide, nitric oxide precursors, or combinations thereof.31. The method of claim 30, wherein the act of applying bacteriacomprises applying the bacteria to the skin between intervals ofbathing.
 32. The method of claim 30, wherein the act of metabolizing anormal constituent of skin secretion comprises metabolizing a componentof perspiration.
 33. The method of claim 31, wherein the act of applyingthe bacteria between intervals of bathing includes applying the bacteriaat an interval of at least about one day.
 34. The method of claim 32,wherein the act of metabolizing comprises metabolizing a component ofperspiration selected from any of urea, nitrites lactic acid, nitrate,salt, iron salts, ammonium salts, and combinations thereof.
 35. Themethod of claim 33, wherein the act of applying the bacteria betweenintervals of bathing comprises applying the bacteria at an interval thatis greater than or equal to about one week.
 36. The method of claim 31,wherein the at of applying the bacteria is applied to a subject wholacks bathing facilities, is immobile, or has a bandage.
 37. The methodof claim 30 wherein the act of applying the bacteria comprises locallyapplying the bacteria to genitalia prior to engagement in a sexual act.38. The method of claim 30, wherein the act of applying the bacteriaincludes locally applying the bacteria to a penis.
 39. The method ofclaim 30, where in the act of applying the bacteria comprises locallyapplying the bacteria to a vagina.
 40. The method of claim 2, furthercomprising applying any of urea, ammonium salts, sodium, potassium,magnesium, calcium, phosphate, chloride, sulfate, trace mineral salts,iron, coppers zinc, cobalt, manganese, molybdenum, buffers, andcombinations thereof to the surface of the subject in an effectiveamount to stimulate the growth of the bacteria.
 41. A preparation to beapplied to a surface of a subject comprising ammonia oxidizing bacteriathat metabolizes perspiration when present, into any of nitric oxide,nitric oxide precursors, or combinations thereof.
 42. The preparation ofclaim 41, wherein the preparation is any of a cosmetic composition, abody deodorant, or an athletic preparation.
 43. The preparation of claim41, wherein the bacteria is selected from any of Nitrosomonas,Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, Nitrosovibrio,and combinations thereof.
 44. The preparation of claim 41, wherein thecomponent of perspiration is selected from any of urea, nitrite, lacticacid, nitrate, salt, iron salts, ammonium salts, and combinationsthereof.
 45. The preparation of claim 42, further comprising at leastone component selected from any of water, mineral oil, coloring agent,perfume, aloe, glycerin, sodium chloride, pH buffers, UV absorbingagents, silicone oil, natural oil, vitamin E, herbal concentrates,Lactic acid, citric acid, talc, clay, calcium carbonate, magnesiumcarbonate, zinc oxide, starch, urea, nitrite, nitrate, iron salts,ammonium salts, and combinations thereof.
 46. The preparation of claim42, wherein the preparation is any of powder, cream, stick, aerosol, orsalve.
 47. The preparation of claim 41, wherein the surface of thesubject is of a human being.
 48. The preparation of claim 41, furthercomprising a component selected from any of water, mineral oil, coloringagent, perfume, aloe, glycerin, Sodium chloride, pH buffers, UVabsorbing agents, silicone oil, natural oils, vitamin E, herbalconcentrates, lactic acid, citric acid, talc, clay, calcium carbonate,magnesium carbonate, zinc oxide, starch, urea, nitrite, lactic acid,nitrate, iron salts, ammonium salts, and combinations thereof.
 49. Thepreparation of claim 41, further comprising: at least one compoundselected from any of urea, ammonium salts, sodium, potassium, magnesium,calcium, phosphate, chloride, sulfate, trace mineral salts, iron,copper, zinc, cobalt, manganese, molybdenum, buffers, and combinationsthereof.
 50. An article of clothing comprising: the article of clothingtreated with bacteria adapted to metabolize any of ammonia, ammoniumsalts, or urea into any of nitric oxide, nitric oxide precursors, orcombinations thereof.
 51. The article of claim 50 wherein the ammonia,ammonium salts, or urea are a component of any of perspiration, urine,feces, blood, wound secretions, menstrual secretions, vaginal;secretions, topically applied mixtures, and combinations thereof. 52.The article of claim 50, wherein the article of clothing is a diaper.53. The article of claim 50, wherein the article is selected from any ofa shoe, sneaker, belt, hat, undergarment, athletic garment, sock, shoeinsert, bandage, face mask, scarf, tampon, and condom.
 54. The articleof claim 50, wherein the bacteria is an ammonia oxidizing bacteria. 55.The article of claim 50, further comprising at least one of urea,nitrite, lactic acid, nitrate, salt, iron salts, ammonium salts, andcombinations thereof.
 56. The article of claim 54, wherein the bacteriais selected from any of Nitrosomonas, Nitrosococcus, Nitrosospira,Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof.